Comparison of intestinal folate carrier clone expressed in IEC-6 cells and in Xenopus oocytes.
We recently identified a cDNA clone from mouse small intestine, which appears to be involved in folate transport when expressed in Xenopus oocytes. The open reading frame of this clone is identical to that of the reduced folate carrier (RFC) (K. H. Dixon, B. C. Lanpher, J. Chiu, K. Kelley, and K. H. Cowan. J. Biol. Chem. 269: 17-20, 1994). The characteristics of this cDNA clone [previously referred to as intestinal folate carrier 1 (IFC-1)] expressed in Xenopus oocytes, however, were found to be different from the characteristics of folate transport in native small intestinal epithelial cells. To further study these differences, we determined the characteristics of RFC when expressed in an intestinal epithelial cell line, IEC-6, and compared the findings to its characteristics when expressed in Xenopus oocytes. RFC was stably transfected into IEC-6 cells by electroporation; its cRNA was microinjected into Xenopus oocytes. Northern blot analysis of poly(A)+ RNA from IEC-6 cells stably transfected with RFC cDNA (IEC-6/RFC) showed a twofold increase in RFC mRNA levels over controls. Similarly, uptake of folic acid and 5-methyltetrahydrofolate (5-MTHF) by IEC-6/RFC was found to be fourfold higher than uptake in control sublines. This increase in folic acid and 5-MTHF uptake was inhibited by treating IEC-6/RFC cells with cholesterol-modified antisense DNA oligonucleotides. The increase in uptake was found to be mainly mediated through an increase in the maximal velocity (Vmax) of the uptake process [the apparent Michaelis-Menten constant (K(m)) also changed (range was 0.31 to 1.56 microM), but no specific trend was seen]. In both IEC-6/RFC and control sublines, the uptake of both folic acid and 5-MTHF displayed 1) pH dependency, with a higher uptake at acidic pH 5.5 compared with pH 7.5, and 2) inhibition to the same extent by both reduced and oxidized folate derivatives. These characteristics are very similar to those seen in native intestinal epithelial cells. In contrast, RFC expressed in Xenopus oocytes showed 1) higher uptake at neutral and alkaline pH 7.5 compared with acidic pH 5.5 and 2) higher sensitivity to reduced compared with oxidized folate derivatives. Results of these studies demonstrate that the characteristics of RFC vary depending on the cell system in which it is expressed. Furthermore, the results may suggest the involvement of cell-or tissue-specific posttranslational modification(s) and/or the existence of an auxiliary protein that may account for the differences in the characteristics of the intestinal RFC when expressed in Xenopus oocytes compared with when expressed in intestinal epithelial cells.
Kumar
C K
CK
Medical Research Service, Veterans Affairs Medical Center, Long Beach 90822, USA.
Nguyen
T T
TT
Gonzales
F B
FB
Said
H M
HM
D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid / Q000494:pharmacology; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid / Q000494:pharmacology; D000818:Animals; D002352:Carrier Proteins / Q000096:biosynthesis / Q000502:physiology*; D002460:Cell Line; D003001:Cloning, Molecular; D018274:Electroporation; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D005493:Folic Acid Antagonists / Q000378:metabolism; D007413:Intestinal Mucosa / Q000502:physiology*; D007421:Intestine, Small; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D051379:Mice; D009838:Oligodeoxyribonucleotides / Q000494:pharmacology; D016376:Oligonucleotides, Antisense / Q000494:pharmacology; D009865:Oocytes / Q000502:physiology*; D011994:Recombinant Proteins / Q000096:biosynthesis; D058978:Reduced Folate Carrier Protein; D014162:Transfection; D014982:Xenopus laevis
D002352:Carrier Proteins; D005493:Folic Acid Antagonists; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D011994:Recombinant Proteins; D058978:Reduced Folate Carrier Protein; C548700:Slc19a1 protein, mouse; C492292:Slc19a2 protein, mouse; D012856:4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfonic Acid; C032418:5,11-methenyltetrahydrohomofolate; D005492:Folic Acid; D017878:4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid